Role of sphingosine kinase and sphingosine-1-phosphate receptor in the liver pathology of mice infected with Plasmodium berghei ANKA

Decreased serum sphingosine 1-phosphate (S1P) has been reported in severe malaria patients, but the expression of receptors and enzymes associated with S1P has not been investigated in the liver of malaria patients. Therefore, this study aimed to investigate the expression of sphingosine kinase (SphK) and S1P receptors (S1PRs) in the liver of malaria-infected mice. C57BL/6 male mice were divided into a control group (n = 10) and a Plasmodium berghei (PbA)-infected group (n = 10). Mice in the malaria group were intraperitoneally injected with 1×106 P. berghei ANKA-infected red blood cells, whereas control mice were intraperitoneally injected with normal saline. Liver tissues were collected on Day 13 of the experiment to evaluate histopathological changes by hematoxylin and eosin staining and to investigate SphK and S1PR expression by immunohistochemistry and real-time PCR. Histological examination of liver tissues from the PbA-infected group revealed sinusoidal dilatation, hemozoin deposition, portal tract inflammation and apoptotic hepatocytes, which were absent in the control group. Immunohistochemical staining showed significant increases in the expression of SphK1 and SphK2 and significant decreases in the expression of S1PR1, S1PR2, and S1PR3 in the endothelium, hepatocytes, and Kupffer cells in liver tissue from the PbA-infected group compared with the control group. Real-time PCR analysis showed the upregulation of SphK1 and the downregulation of S1PR1, S1PR2, and S1PR3 in the liver in the PbA-infected group compared with the control group. In conclusion, this study demonstrates for the first time that SphK1 mRNA expression is upregulated and that S1PR1, S1PR2, and S1PR3 expression is decreased in the liver tissue of PbA-infected mice. Our findings suggest that the decreased levels of S1PR1, S1PR2, and S1PR3 might play an important role in liver injury during malaria infection.

3. Not all abbreviations are adequately explained (e.g., line 110)please correct. Response: Thank you for this valuable suggestion. This abbreviation refers to the names and surnames of two expert pathologists. In response to this comment and to avoid confusion, the abbreviations have been deleted. Please see page 6, line 114. Figure 2 is of low quality, and it is impossible to evaluate the staining results. Response: Figure 2 has been revised as suggested. The new image has a resolution of 600 dpi. Please see the attached file.

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5. More appropriate for expression data would be boxplot and not the histogram. Also, Figure  3 is incorrectly referenced in the text as Figure 2. Response: Thank you very much for your comment. In response to this comment, we have revised the graph and corrected the reference as suggested. Please see Figure 3 and page 11 (lines 212 and 214). 6. Line 208: Authors contradict themselves. First, they write that there are differences in mRNA level of SphK1, to add later that it did not change… Moreover, S1PR1 and S1PR2 mRNA expression are lower only by 30-40% when compared with appropriate controls, so it is not a 3-fold difference. Not to mention, these results are not statistically significant due to high variability between individual animals. Response: First, we apologize for the error that you noted. In the later instance mentioned regarding SphK1, we were referring to SphK2 mRNA levels. Therefore, we altered the text to indicate the correct marker in response to this criticism. Please see page 11, line 211. Second, we double-checked and reanalyzed the laboratory results. As you mentioned, we discovered that the mRNA expression levels of S1PR1 and S1PR2 were reduced by 30-40% in the PbAinfected group. However, a significant decrease in S1PR3 mRNA expression was observed in the PbA-infected group compared with the control group. As a result, the text and graph have been updated. Please see lines 211-214 on page 11 and Figure 3.
7. Line 251. Downregulation of mRNA expression was only shown for S1PR3, so writing that it affects all investigated receptors is an overstatement. Response: In response to comment 6, we double-checked the results. Except for S1PR3, all the receptors had lower mRNA expression in the PbA-infected group, but the differences were not significant. 8. It is not unusual to not see differences at the mRNA level but notice the differences at the protein level (and vice versa), and authors should discuss this in their discussion. Moreover, it would be beneficial for the readers if possible explanation or more detailed information about receptors processing/degradation could be included in the discussion. Response: We have updated the discussion on the interaction between SphKs and S1PRs. Please see page 15, lines 293-298.
Reviewer #2: The study tested levels of SphK1 mRNA expression in liver tissues of mice infected with malaria pathogens. Authors also found that S1PR1, S1PR2, and S1PR3 expressions are decreased in the liver tissue of Plasmodium berghei-infected mice. Data suggests that the decreased levels of S1PR1, S1PR2, and S1PR3 may be involved in liver injury during malaria infection. The study is very interesting. However, there are several issues to address. The manuscript should be improved/amended. 1. Text requires proper English language editing. Response: A revised manuscript was thoroughly edited by highly qualified native Englishspeaking editors at American Journal Experts.
2. Figure 2 resolution is poor. Author should provide images with higher magnification and/or better resolution. Response: Figure 2 has been revised as suggested. The new image has a resolution of 600 dpi. Please see the attached file.